The NZB mouse model of autoimmune hemolytic anemia was one of the first animal models of autoimmunity and has been important in contributing to our understanding of genetic control of anti-self responses and some of the cell interactions involved in these responses. The 100% incidence of disease in the NZB mice and their ability to control this response until middle age allows study of the model prior to, as well as after initiation of specific autoantibody production. Yet, the mechanisms of induction of the anti-erythrocyte autoantibodies in this mouse model are not known. Cellular studies of the controlling elements in this response have so far been limited by the use of an intact erythrocyte as the target antigen, for which assays of specific responses are complex and not very sensitive for low affinity antibodies. The use of target peptide MRBC epitopes could simplify and enhance the sensitivity of assays for this autoimmune response, making possible mechanistic studies of this autoantibody reaction that have not so far been approachable. A major portion of the pathogenic autoantibodies produced in these mice bind to erythrocyte band 3, however the actual target epitopes have not been defined, and band 3, as an integral membrane protein is difficult to obtain with structural integrity. Therefore, we propose a pilot study to focus on identifying the specific band 3 target epitopes of the NZB autoantibodies and then using these epitopes to develop assays that will be usable to detect individual autoantibody responses to each target epitope. Linear epitopes will be identified by testing selected peptide sequences deduced from DNA sequences of the extracellular loops of the erythrocyte band 3 for binding to a panel of pathogenic NZB monoclonal autoantibodies as well as to polyclonal autoantibodies extracted from autoimmune NZB mice. Target conformational epitopes of band 3 will be identified as mimotopes using a phage display approach for epitope discovery. ELISPOT assays for epitope-specific autoantibody secreting cells (AFC) will be developed using the selected target peptide epitopes as detection probes for secreted antibody. These assays will then be used to determine the importance of identified band 3 epitopes or mimotopes at each stage of NZB disease, focusing on the earliest stage identifiable to determine which epitope target(s) is/are involved in the initiation of this autoimmunity. This pilot study should make possible future investigations into the regulation of each epitope-specific autoantibody response in normal as well as pre- autoimmune and actively autoimmune NZB mice and into potential causes of the failure of these regulatory mechanisms in autoimmune NZB mice. The ultimate goal of these investigations into regulatory mechanisms that control the anti-MRBC response in NZB mice is to open up and test therapeutic opportunities for human autoimmune disease. NZB mice provide an animal model of human autoimmune disease in which anti-self erythrocyte (MRBC) antibodies are spontaneously produced and detectable in mid-life. The goal of the proposed work is to identify the MRBC peptide targets of the anti-MRBC autoantibodies made by these mice and to use these target peptides to develop assays for the autoreactive cells. Such assays will facilitate studies of the mechanisms of control versus induction of these autoantibody responses that will lead ultimately to therapeutic interventions in human autoimmune disease. [unreadable] [unreadable] [unreadable]